Test device and method for colored particle immunoassay

ABSTRACT

Disclosed is a test cell and a method for detection of a preselected ligand in a liquid sample such as a body fluid. The test cell includes an elongate outer casing which houses an interior permeable material capable of transporting an aqueous solution and defining a sample inlet, a test volume, and a reservoir volume. The reservoir volume is disposed in a section of the test cell spaced apart from the inlet and is filled with sorbent material. The reservoir acts to receive liquid transported along a flow path defined by the permeable material and extending from the inlet and through the test volume. In the test volume is a test site which includes a first protein having a binding site specific to a first epitope of the ligand immobilized in fluid communication with the flow path. The test site can be observed through a window of the casing.

BACKGROUND OF THE INVENTION

This invention relates to assays for ligands, e.g., antigens, in aliquid sample such as a body fluid. More particularly, the inventionrelates to a method and apparatus for the detection of a ligand in abody fluid such as urine using a conjugate comprising colored particlesand a novel flow-through test cell.

Many types of ligand-receptor assays have been used to detect thepresence of various substances, often generally called ligands, in bodyfluids such as urine. These assays involve antigen antibody reactions,synthetic conjugates comprising radioactive, enzymatic, fluorescent, orvisually observable metal sol tags, and specially designed reactorchambers. In all these assays, there is a receptor, e.g., an antibody,which is specific for the selected ligand or antigen, and a means fordetecting the presence, and often the amount, of the ligand-receptorreaction product. Most current tests are designed to make a quantitativedetermination, but in many circumstances all that is required is apositive/negative indication. Examples of such qualitative assaysinclude blood typing and most types of urinalysis. For these tests,visually observable indicia such as the presence of agglutination or acolor changes are preferred.

Even the positive/negative assays must be very sensitive because of theoften small concentration of the ligand of interest in the test fluid.False positives can also be troublesome, particularly with agglutinationand other rapid detection methods such as dipstick and color changetests. Because of these problems, sandwich assays and other sensitivedetection methods which use metal sols or other types of coloredparticles have been developed. These techniques have not solved all ofthe problems encountered in these rapid detection methods.

It is an object of this invention to provide a rapid, sensitive methodfor detecting ligands in body fluids. Another object is to provide anassay which has high sensitivity and fewer false positives thanconventional assays. A further object is to provide a test cell fordetection of low levels of ligands in body fluids. Another object is toprovide an assay system which involves a minimal number of proceduralsteps, and yields reliable results even when used by untrained persons.

These and other objects and features of the invention will be apparentfrom the following description, drawing, and claims.

SUMMARY OF THE INVENTION

The invention features a method and test cell for the detection of apreselected ligand in a liquid sample such as a body fluid.

The test cell useful in the practice of the invention has an elongateouter casing which houses an interior permeable material, e.g., glassfiber, capable of transporting an aqueous solution by capillary action,wicking, or simple wetting. The casing defines a sample inlet, andinterior regions which, for ease of description, can be designated as atest volume and a reservoir volume. The reservoir volume is disposed ina section of the test cell spaced apart from the inlet, and preferablyis filled with sorbent material. The reservoir acts to receive liquidtransported along a flow path defined by the permeable material andextending from the inlet and through the test volume. In the test volumeis a test site comprising a first protein having a binding site specificto a first epitope of the ligand immobilized in fluid communication withthe flow path, e.g., bound to the permeable material or to latexparticles entrapped in or bonded to the permeable material. A windowsuch as a hole or transparent section of the casing permits observationsof the test site through the casing wall.

In a preferred embodiment, the flow path is restricted or narrowed inthe test area, thereby channeling and concentrating fluid-flow intocontact with the test site. It is also preferred that the test cellinclude a solution filtering means disposed in the flow path between thesample inlet and the test site. The filtration means can comprise aseparate, conventional filter element disposed within the casing of thetest cell in fluid communication with the permeable material definingthe flow path, but preferably is defined simply by a portion of thepermeable material itself. The provision of such a filtration means inthe test cell has the effect of removing by entrapment from impuresamples, such as urine samples, a portion of the particulates andnonspecific interfering factors which sometimes cause false positivereadings.

The method of the invention requires the use of a conjugate comprising asecond protein bound to colored particles such as a metal sol orcolloid, preferably gold. The conjugate can take two distinct forms,depending on whether the assay is designed to exploit the “sandwich” or“competitive” technique.

In the case of the sandwich technique, the second protein comprises asite which binds to a second epitope on the ligand. This type ofconjugate reacts with the ligand to form a complex in the liquid sample.The complex is detected by visual observation of color development atthe test site in the test cell. At the test site, the ligand bound withthe conjugate reacts with the immobilized first binding protein to forma “sandwich” of the first protein, ligand, second protein, and coloredparticles. This sandwich complex is progressively produced at the testsite-as sample continuously passes by, filling the reservoir. As moreand more conjugate is immobilized, the colored particles aggregate atthe test site and become visible through the window, indicating thepresence of ligand in the liquid sample.

In the case of the competitive technique, the second protein binds withthe first protein in competition with the ligand. The second proteincomprises, for example, an authentic sample of the ligand or a fractionthereof which has comparable affinity for the first protein. As theliquid sample is transported in contact with the test site, ligand, ifany, and the conjugate compete for sites of attachment to the firstprotein. If no ligand is present, colored particles aggregate at thetest site, and the presence of color indicates the absence of detectablelevels of ligand in the sample. If ligand is present, the amount ofconjugate which binds at the test site is reduced, and no color, or apaler color, develops.

In one embodiment of the invention, the test liquid is mixed with theconjugate outside the test cell. In another embodiment, the conjugate isdisposed in freeze-dried or other preserved form on the permeablematerial between the inlet and the test site, and the sample liquidresolubilizes the conjugate as it passes along the flow path.

Color development at the test site may be compared with the color of oneor more standards or internal controls to determine whether thedevelopment of color is a true indication of the presence or absence ofthe ligand, or an artifact caused by nonspecific sorption.

In one embodiment employing the sandwich technique, the standardconsists of a negative control site, preferably disposed adjacent thetest site, and visible through a second window proximate the first. Thenegative control site preferably is prepared identically to the testsite, except immobilization of the first binding protein is omitted.Therefore, although the conjugate will reach the control site, itaggregates due only to non-specific binding. If the test site is notappreciably more intense in color than the control site, the assay isconsidered negative.

In another embodiment, the assay and test cell may include a positivecontrol. Thus, when exploiting the sandwich technique, the cell may havean authentic sample of the ligand immobilized at a control site. If nocolor develops at this control site, the assay is consideredinconclusive. When exploiting the competitive technique, the developmentof color at the positive control site means the assay results areinconclusive.

Broadly, the method of the invention involves the use of a test cell ofthe type described above to achieve an easily readable, sensitive,reproducible indication of the presence of a ligand, e.g., humanchorionic gonadotropin (hCG), in a test sample such as a human urinesample. The method involves the step of transporting the sample and aconjugate comprising a protein bound to a metal sol or other coloredparticle along a flow path and in contact with a test site comprisingimmobilized binding protein specific to an epitope of the ligand, andpreferably also in contact with a control site. Preferably, the coloredparticle comprises a gold sol; the flow path in the region of the testsite is reduced in cross-section relative to other parts of the flowpath; the sample is passed through a filtration means after it entersthe test cell but before it contacts the test site; and the test sitecomprises latex particles entrapped or otherwise fixed in the flow pathhaving the immobilized protein on their surface. In the practice of theprocess, either the conjugate is premixed with the sample, or theconjugate is disposed in preserved form, e.g., lyophilized, in the flowpath between the inlet and the test site. In either case, placement ofthe test cell in the sample, or application of the sample to the inlet,initiates flow, and the result is read by observing color development athe test site, or by comparing the color of the test site and controlsite.

The use of the colored particle detection system in combination with thefiltration means, the concentrating effect of flow of the sample, andthe ease of comparison between the colors of the test and control sites,together enable construction of a family of extremely sensitive assaysystems which minimize false positives and can be used effectively byuntrained persons.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a cut-away, schematic, top view of an embodiment of a testcell useful in explaining the test cell and process of the invention;

FIG. 2 is a cross-sectional side view of the test cell of FIG. 1;

FIG. 3 is a perspective view of a currently preferred test cellconstructed in accordance with the invention;

FIG. 4A is a cross-sectional, top view of the test cell of FIG. 3;

FIG. 4B is a cross-sectional, side view of the test cell of FIG. 3 takenat line 4B-4B of FIG. 4A;

FIG. 5 is a cross sectional view of the cell of FIG. 3 taken at line 5-5of FIG. 4B; and

FIG. 6 is a perspective view of another embodiment of a test cellconstructed in accordance with the invention.

Like reference characters in the respective drawn figures indicatecorresponding parts.

DESCRIPTION

The invention provides a test cell for conducting a sandwich orcompetitive immunoassay, and a process which utilizes the test cell anda conjugate comprising colored particles. As disclosed below, variousfeatures of the process and test cell of the invention cooperate toenable untrained personnel reliably to assay a liquid sample for thepresence of extremely small quantities of a particular ligand whileavoiding false positives and simplifying test procedures. The inventionis ideal for use in over-the-counter assay test kits which will enable aconsumer to self diagnose, for example, pregnancy, venereal disease, andother disease, infection, or clinical abnormality which results in thepresence of an antigenic marker substance in a body fluid, includingdetermination of the presence of metabolites of drugs or toxins. Theassay process and the cell are engineered specifically to detect thepresence of a preselected individual ligand present in a body or otherfluids.

Broadly, the test cell and process of the invention can be used todetect any ligand which has heretofore been assayed using knownimmunoassay procedures, or known to be detectable by such procedures,using polyclonal or monoclonal antibodies or other proteins comprisingbinding sites for ligands. Various specific assay protocols, reagents,and analytes useful in the practice of the invention are known per se,see, e.g., U.S. Pat. No. 4,313,734, columns 4-18, and U.S. Pat. No.4,366,241, columns 5-40.

The combination of features believed to be responsible for the excellentsensitivity and reproducibility of assays constructed in accordance withthe invention is the use of the novel test cell which serves toconcentrate ligand from a test sample at a test site in the cell, andthe use of a metal sol or other colored particle as a marker systemwhich permits direct visual observation of color development. Falsepositives are reduced while maintaining excellent sensitivity byincluding in the test cell a negative control or control site whosecolor is compared with the test site, and by including a filtrationmeans which limits the introduction to the test site of contaminantsfrom the sample.

The assay is conducted by simply placing the inlet of the test cell incontact with a liquid test sample. One then merely waits for the testsample to pass through the cell and into reactive contact with the testsite (and optionally one or more control sites) visible through a windowor windows in the cell's exterior casing. In one embodiment, theconjugate is mixed with the sample and incubated briefly before the testcell is inserted. In another embodiment, the conjugate is disposed inpreserved form in the flow path within the cell. If the ligand ispresent in the sample, it passes through the inlet and the interior ofthe cell along the flow path past the test and control sites, where, inthe sandwich embodiment, it reacts with immobilized binding protein,e.g., antibody, at the test site, and perhaps also non-specifically atthe control site. A “sandwich” forms at the test site comprisingimmobilized binding protein-ligand binding protein-colored particle. Thepresence of the sandwich complex and thus the ligand is indicated by thedevelopment of color caused by aggregation of the metal sol particles atthe test site. A deeper color at the test site than at the negativecontrol site is a positive indication of the presence of the ligand.

By providing a reservoir of sorbent material disposed beyond the testand control sites, a relatively large volume of the test liquid and anyligand it contains can be drawn through the test area to aidsensitivity. Optionally, the region of the flow path in the test celldefining the test and control sites is restricted in cross-sectionalarea relative to other regions of the flow path. This feature produces a“bottle-neck” effect wherein all ligand in the entire volume of sorbedsample must pass through the restricted flow area immediately about thetest site where it will be immobilized by reaction with binding protein.

From the foregoing, it will be apparent that the success of the testprocedure is dependent on ligand present in the sample reacting with theconjugate, or on reproducible competition between the ligand and theconjugate for sites of attachment at the test site. In accordance withthe invention, as noted above, the assays can be conducted by premixingthe conjugate with the liquid sample prior to introduction into theelongate test cell. Alternatively, the conjugate may be disposed inpreserved form, e.g., freeze-dried, in the flow path within the testcell upstream of the test and control sites. In this case, the cell isplaced directly in the liquid sample solution without premising. Ligand,if any, passing up through the cell and entrained within the liquidmoves into contact with the conjugate forming an immune complex orinitiating competition in situ as flow continues. This latter techniquehas the advantage that it eliminates a manipulative step in the assayprocedure, and accordingly a possible source of error.

Referring to the drawing, FIGS. 1 and 2. illustrate schematically anembodiment of a test cell 5 constructed in accordance with the inventionuseful in explaining its principles of construction. It comprises anouter, molded casing 10 which defines a hollow, elongate enclosurefilled with a permeable, sorbent material 12. Casing 10 also defines atest liquid inlet 14 and a pair of circular openings 16, 18 comprisingwindows through which sorbent material 12 is visible.

Sorbent material 12 and the interior of casing 10 together define a flowpath passing generally from left to right in FIGS. 1 and 2. When thetest cell is placed with inlet 14 disposed within or otherwise incontact with a liquid sample, the liquid is transported by capillaryaction, wicking, or simple wetting along the flow path through upstreamflow section 20, test volume 22, and into reservoir volume 24, generallyas depicted by the arrows. The flow section 20 of the flow path disposedinwardly of the inlet 14 serves as a filter which can remove from impuretest samples particulate matter and interfering factors. The provisionsof such a filtration means 20 downstream of the inlet 14 is believed tocontribute to the success of the system and its ability to avoid falsepositives.

Disposed within sorbent material 12 is a band 26 of dehydratedconjugate, e.g., antibody-metal sol. As the liquid sample moves pastband 26, the conjugate is entrained in the liquid, reconstituted, andreacts or competes with ligand, if present, dissolved in the liquidsample. Of course, conjugate band 26 may be eliminated, and theconjugate added to the test liquid prior to introduction of the cell 5as previously noted.

Within the volume of sorbent material 12 disposed directly beneathcircular openings 16 and 18 in casing 10 is disposed, respectively,control site 16′ and test site 18′. In the drawing, the control and testsite are illustrated as being disposed serially along the flow path.Alternatively, the control and test site or sites may be disposed sideby side or in other spacial relationships.

Test site 18′ comprises a preselected quantity of antibody against anepitope of the ligand to be detected immobilized in place within theflow path. Its detailed chemical structure can vary widely. Control site16′ is preferably identical in size and chemical makeup to test site18′, excepting that the immobilized antibody present at the test site18′ is omitted at the control site 16′. Thus, any nonspecificaggregation of, e.g., ligand-conjugate or free conjugate, which occursat test site 18′ also will occur at control site 16′. A deeper color attest site 18′ as compared with control site 16′ is a positive indicationof ligand in the sample in the sandwich assay.

The invention is not limited by the precise nature of the test site 18′and corresponding control site 16′, and in fact, control site 16′ may beentirely eliminated if a reduction in sensitivity can be tolerated.Generally, antibody or other binding protein may be immobilized at testsite 18′ using adsorption, absorption, or ionic or covalent coupling, inaccordance with methods known per se. A currently preferred formulationfor test site 18′ is to immobilize monoclonal antibody against anepitope of the ligand on latex beads, and then to entrap or otherwiselink the beads in sorbent material 12 at region 18′. Control site 16′ isfabricated identically, except that the latex beads contain non specificimmunoglobulin, e.g., immunoglobulin from bleedings from an animal thathas not been immunized.

Disposed beyond test volume 22 is a reservoir volume 24 comprising arelatively large mass of sorbent or supersorbent material. The purposeof reservoir volume 24 is to assure that a reasonably large amount oftest liquid is drawn through test volume 22. Increasing the volume ofreservoir 24 can have the effect of increasing the sensitivity of theassay procedure, as it results in an increase in the amount of ligandpassing through the test area 22. Suitable sorbents include commercialmaterials of the type available, for example, from The Dow ChemicalCompany of Midland, Mich., and the Chemical division of AmericanColloid, Arlington Heights, Ill. These materials can absorb many timestheir weight in water and are commonly used in disposable diapers. Theycomprise lightly crosslinked polyacrylate salts, typically alkali metalsalts.

Polyclonal antisera and indeed monoclonal antibodies or fractionsthereof having specific binding properties and high affinity forvirtually any antigenic substance are known and commercially availableor can be produced from stable cell lines using well known cell fusionand screening techniques. The literature is replete with proteinimmobilization protocols. See, for example, Laboratory Techniques inBiochemistry and Molecular Biology, Tijssen, Vol. 15, Practice andTheory of Enzyme immunoassays, chapter 13, The Immobilization ofImmunoreactants on Solid Phases, pp. 297-328, and the references citedtherein.

Metal sols and other types of colored particles useful as markersubstances in immunoassay procedures are also known per se. See, forexample, U.S. Pat. No. 4,313,734, Feb. 2, 1982, to Leuvering, thedisclosure of which is incorporated herein by reference. For details andengineering principles involved in the synthesis of colored particleconjugates see Horisberger, Evaluation of Colloidal Gold as aCytochromic Marker for Transmission and Scanning Electron Microscopy,Biol. Cellulaire, 36, 253-258 (1979); Leuvering et al, Sol ParticleImmunoassay, J. Immunoassay 1 (1), 77-91 (1980), and Frens, ControlledNucleation for the Regulation of the Particle Size in Monodisperse GoldSuspensions, Nature, Physical Science, 241, pp. 20-22 (1973).

The cell can take various forms. It will usually comprise an elongatecasing comprising interfitting parts made of polyvinyl chloride,polypropylene, or other thermoplastic resin. Its interior flow path willcontain a relatively inert material or a combination of materialssuitable for transporting the liquid. In some circumstances it may bepreferable to use a material of higher sorptivity as the reservoir,promoting the flow of liquid, and a different material for remainingportions of the flow path.

From the foregoing it should be apparent that the advantages inreproducibility, sensitivity, and avoidance of false positives of assaysystems constructed in accordance with the invention are traceable to acombination of features of the invention. In use, the test cell of theinvention and the metal sol particles used as a marker togethercooperate to result in an increase in color intensity progressively asligand complexed with conjugate is trapped at the test site by theimmobilized binding protein. This approach can be utilized to designassays and test cells for essentially any antigenic material.

The invention will be further understood from the following non-limitingexamples.

EXAMPLE 1

The currently preferred test device embodying the invention is shown inFIGS. 3, 4A, 4B, and 5. A modification of the device depicted in FIG. 3is shown in FIG. 6, and includes a second control site 19 in addition tocontrol site 16′ and test site 18′, as well as a stand 21 useful formaintaining the test cell in an incline position with the reservoirdownhill. When a test sample is applied to inlet 14, gravity as well assorption aids in transporting the sample along the flow path.

As shown in FIGS. 3, 4A, 4B, and 5, the preferred test cell of theinvention differs from the exemplary device discussed above and shown inFIGS. 1 and 2 in certain of its more specific internal features.Specifically, the casing comprises a pair of interfitting polymericparts including a U-shaped top part 10 which, when the device isassembled, interfits with lower part 10′. Top and bottom parts 10 and10′ may be connected through a hinge region 11. The bottom section 10′defines a pair of channels 28 above which is disposed a strip of glassfiber paper 13 (available commercially from Eaton Dikeman, Grade 111, orWhatman, Grade GFA). Test liquid applied through inlet 14 soaks alongthe paper strip 13 which defines the flow path and a filtering meansregion 20, as well as a positive control site 16′ and test site 18′visible through windows 16 and 18 consisting of openings through uppermating member 10. The paper strip 13 overlaps into reservoir volume 24,which is defined by a cavity between the interfitting top and bottommating members 10 and 10′. The cavity in this case is filled withsorbent blotting paper 12 comprising the sorbent reservoir. A suitablepaper is sold as Grade 12A absorbent paper, a cellulose productavailable from Schleicher and Schuell. In one preferred embodiment, thedimensions of the glass fiber paper 13 were approximately one quarterinch by three inches, and those of the absorbent material 12approximately two inches by five thirty seconds of an inch on each side.A number of these devices were produced and further treated to adaptthem to detect pregnancy by assay of urine.

Test site 18′ in each device was fabricated as a spot within fiber paper13 using the following technique. Latex beads available commercially andcomprising polystyrene particles 0.3 micron in diameter were passivelycoated with purified rabbit anti-human chorionic gonadotropin. Thepolyclonal antibody was purified using conventional techniques frombleedings of a rabbit previously immunized with human chorionicgonadotropin in a manner know per se. Equal parts of a latex (0.6% byweight) having a continuous phase of glycine buffer, pH=8.3, and a 1mg/ml antibody solution in the same buffer were mixed and incubated at37° C. 15 microliters of this solution, comprising approximately 0.6%solids, were added, one drop at a time, to the glass fiber paper 13 toproduce spot 18′ after the devices had been assembled. The spots werethen allowed to dry at 37° C. The control site 16′ was producedidentically to the test site disclosed immediately above, excepting thatrabbit polyclonal non-immune gamma globulin was used in place of theanti-hCG gamma globulin.

Metal sol particles were prepared in accordance with the method ofFrens, Controlled Nucleation for the Regulation of the Particle Size inMono Dispersed Gold Solutions (1973), supra. Briefly, the gold sol wasprepared by reducing a 4% solution of gold chloride with 1% sodiumcitrate to produce gold particles of approximately 18 nm in diameter.The particles were made immunochemically reactive by admixture with amonoclonal antibody specific for human chorionic gonadotropin with nodetectable cross-reactivity with human leutinizing hormone. The antibodywas purchased from Charles River Labs, and is produced using standardtechniques including purification from ascites using HPLC ion exchangechromatography. It was added to the gold sol as a 10 ug/ml solution inborate buffer, pH-6. The bound antibody fraction is separated from thefree fraction by either density centrifugation or gel filtrationchromatography. Additional details of the currently preferred procedurefor making the antibody sol conjugate are disclosed by Leuvering et al,J. Immunoassay (1980) supra. Individual batches of the latex and theconjugate are titrated to optimize activity so that a suitable amount oflatex is applied to the test site and a suitable amount of conjugate isused in conducting the test.

Test Protocol

To a 10×50 mm test tube of lyophilized gold sol antibody conjugate isadded 0.5 ml urine sample containing a known quantities of hCG. Thesamples comprised hCG standards purchased from Sigma Chemical Companydiluted in processed, hCG negative urine. The contents of the tube aremixed by shaking in a horizontal motion until the lyophilized antibodyis dissolved. The device depicted in FIGS. 3-5 is then inserted into thetube, and the results are read after the entire fluid volume has beenabsorbed.

The results of this qualitative procedure follows: Color of Color of mIUhCG Control Spot Reagent Spot 0 grey grey 25 grey pink hue 50 grey pink100 grey rose 150 grey rose >150 grey dark rose

The pink color clearly visible at 50 mIU of human chorionic gonadotropinmeans that the test can detect pregnancy one day after a missedmenstrual period. In initial stages of testing, approximately 5 negativesamples from various sources have been run with no false positives oreven border-line cases. It is anticipated that the commercial devicewill have less than 1% false positives.

Non-limiting examples of materials which may be assayed in accordancewith the invention in addition to the human chorionic gonadotropin notedabove include human leutinizing hormone, progesterone, estrogen, andstreptococcus.

Other embodiments are within the following claims.

1. A method of detecting a ligand in a liquid sample, the methodcomprising the steps of: A. transporting along a flow path in a testcell a solution, including a liquid sample suspected to contain a ligandand a conjugate, into contact with a test site visible through a windowin a wall of said test cell, said test site having immobilized thereon afirst protein having a binding site specific to a first epitope on theligand, said conjugate comprising colored particles coupled to a secondprotein selected from the group consisting of proteins having a bindingsite specific to a second epitope on the ligand and proteins which bindwith said first protein in competition with the ligand, and B.continuing transport of said solution to progressively produce at saidtest site a complex comprising said ligand for a time sufficient tovisually determine through said window whether a color is developed atsaid test site:
 2. The method of claim 1 wherein said test cellcomprises a filtration means for filtering said liquid sample, saidmethod comprising the additional step of transporting the sample throughsaid filtration means before said sample contacts said test site.
 3. Themethod of claim 1 wherein the cross-sectional area of said flow path isrestricted about said test site whereby ligand is localized at said testsite during flow of solution thereby.
 4. The method of claim 1comprising the additional steps of transporting said solution intocontact with a control site visible through a window in a wall of saidtest cell and comparing the color of said test site and control site. 5.The method of claim 4 wherein said control site comprises a negativecontrol site free of said first protein.
 6. The method of claim 4wherein said control site comprises a positive control site havingimmobilized thereon an authentic sample of said ligand.
 7. The method ofclaim 1 comprising the step of mixing said conjugate with said liquidsample prior to step A.
 8. The method of claim 1 wherein said conjugateis disposed in said flow path, said method comprising the additionalstep of transporting said liquid into solubilizing contact with saidconjugate prior to contact with said test site.
 9. The method of claim 1wherein said first and second proteins comprise antibodies and at leastone of said proteins is a monoclonal antibody.
 10. The method of claim 1wherein said first protein has a binding site specific to an epitope ofhuman chorionic gonadotropin.
 11. The method of claim 1 wherein saidfirst protein has a binding site specific to an epitope of humanprogesterone.
 12. The method of claim 1 wherein said second protein hasa binding site specific to a second epitope on the ligand, and when saidsample contains said ligand, the complex produced in step B comprisessaid ligand bound to both said first and second proteins, and color isproduced by aggregation of said colored particles at said test site. 13.The method of claim 1 wherein said second protein binds with said firstprotein in competition with the ligand, and when said sample containssaid ligand, the complex produced in Step B comprises said ligand boundto said first protein, and when said sample is free of said ligand, thecomplex produced in step B comprises said conjugate bound to said firstprotein, and color is produced by aggregation of said colored particlesat said test site.
 14. A test cell for detecting a ligand in a liquidsample, the test cell comprising an elongate casing for housing apermeable material and defining a liquid sample inlet, a reservoirvolume, a test volume interposed between said inlet and reservoirvolume, and a window through said casing at said test volume, permeablematerial capable of transporting an aqueous solution disposed withinsaid casing and defining a flow path extending from said sample inletthrough said test volume and into communication with said reservoirvolume, a first protein having a binding site specific to a firstepitope on said ligand, said first protein being immobilized at a testsite, disposed within said test volume in fluid communication with saidflow path and visible through said window, and a sorbent material insaid reservoir volume for drawing liquid sample along said flow path andinto contact with said test site.
 15. The cell of claim 14 furthercomprising a liquid sample filtering means disposed in said flow pathbetween said inlet and said test site.
 16. The cell of claim 15 whereinsaid filtering means is defined by a portion of said permeable material.17. The cell of claim 14 wherein the cross sectional area of said flowpath is restricted about said test site so that ligand in liquid passingtherealong is localized at said test site.
 18. The cell of claim 14wherein said casing defines a second window through said casing and saidcell further comprises a control site in fluid communication with saidflow path visible through said second window.
 19. The cell of claim 18wherein said control site comprises a negative control site free of saidfirst protein.
 20. The cell of claim 19 wherein said control sitecomprises latex particles disposed in contact with said permeablematerial.
 21. The cell of claim 18 wherein said control site comprises apositive control site having immobilized thereon an authentic sample ofsaid ligand.
 22. The cell of claim 14 further comprising a conjugatedisposed in said flow path between said test site and said inlet; saidconjugate comprising colored particles coupled to a second proteinselected from the group consisting of proteins having a binding sitespecific to a second epitope on the ligand, and proteins which bind withsaid first protein in competition with the ligand.
 23. The cell of claim14 wherein said test site comprises an antibody fixed to latex particlesdisposed in contact with said permeable material.
 24. The cell of claim14 wherein said first protein binds with an epitope of human chorionicgonadotropin.
 25. The cell of claim 14 wherein said first protein bindswith an epitope of human progesterone.
 26. The cell of claim 22 whereinat least one of said first and second proteins is a monoclonal antibody.